Neurofilaments are known to play a central role in the etiology of a number of human neurodegenerative disorders, most notably motor neuron disease and giant axonal neuropathy. These disorders are characterized by massive accumulations of neurofilaments in the axons of affected neurons, forming giant balloon-like swellings and leading to axonal degeneration. The accumulation of neurofilaments in these diseases is thought to be caused by changes in the mechanisms of slow axonal transport which move cytoskeletal proteins along axons. The mechanism of slow axonal transport is poorly understood and controversial. The principal issue concerns the site of assembly of cytoskeletal proteins and the form in which they move. In the case of neurofilament proteins, considerable evidence suggests that the cell body is a principal site of assembly and that these proteins are transported along the axon as assembled polymers. Alternatively, it has also been proposed that cytoskeletal proteins are transported along axons in a non- polymeric form and that the principal site of assembly is at the axon tip. To test these hypotheses, the assembly and transport of neurofilament proteins will be investigated in cultured neurons using immunofluorescence microscopy and microinjection techniques, in conjunction with quantitative digital image analysis. To identify the sites of assembly of neurofilament proteins, bovine low- molecular weight neurofilament protein (NF-L) will be microinjected into cultured rat neurons and the assembly of the injected protein will be visualized using species-specific monoclonal antibodies. In addition, the sites of assembly of newly synthesized high-molecular weight neurofilament protein (NF-H) will be identified in cultured neurons by taking advantage of its delayed expression during axon outgrowth. To visualize the axonal transport of neurofilaments, Fab fragments of neurofilament-specific antibodies will be microinjected into cultured neurons and the movement of the bound antibodies will be analyzed by immunofluorescence microscopy. To visualize the transport of neurofilament proteins regardless of the form in which they move, the accumulation of transported proteins will be investigated at an experimentally applied axonal constriction. These studies will yield important new information on the assembly and axonal transport of neurofilaments and will lay the groundwork for future studies on the mechanisms of slow axonal transport in cultured neurons, and on the mechanisms that cause neurofilaments to accumulate in axons. An understanding of these mechanisms will shed new light on the etiology of neurofilamentous neuropathies.